Ortho microscope

Renal tissue samples were collected from mice and were fixed in 4% paraformaldehyde, embedded in paraffin and 4 µm thick serial sections were obtained. Then, the sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H & E) in order to assess histopathological kidney injury and Sirus-red and Masson’s trichrome staining for observing collagen deposition. On the basis of the procedure described in previous research, immunohistochemical staining of RCAN1.4 was performed [48 (link)]. The following primary antibody of rabbit anti-RCAN1 (1:200 dilution, pH 8.0, Sigma-Aldrich, USA) was used. The stained tissues were viewed by Ortho microscope (OLYMPUS, Tokyo, Japan).

Kidney samples were placed in 4% paraformaldehyde, followed by being segmented at a thickness of 4 μm from paraffin-embedded tissues. Then hematoxylin and eosin (H&E) were carried out to assess pathological renal injury, and Masson’s trichrome staining and Sirius Red staining to observe collagen deposition. Renal tubule injury severity was determined with five grades (0–4): 0, no obviously visible injury; 1, injury <25%; 2, injury 25–50%; 3, injury 50–75%; and 4, injury >75% [40 (link)–42 (link)].
As for immunohistochemical staining, antibodies, included anti-RCAN1 (1:200, D6694, Sigma-Aldrich), anti-YY1 (1:200, ab109228, Abcam), anti-E-cad (1:100, #40860, SAB), anti-TNF-α (1:200, ab6671, Abcam), anti-α-SMA (1:100, 14395-1-AP, Proteintech), were employed to incubate kidney section, followed by an appropriate secondary antibody. The stained samples were observed using an Ortho microscope (OLYMPUS, Tokyo, Japan).